The increasing demand on the one hand and depleting fossil fuels on the other, as energy sources has necessitated the development of alternative sources of energy. The production of renewable biofuels using naturally abundant lignocellulosic biomass such as agricultural waste, forestry waste and municipal waste will reduce society's dependence on fossil fuels. Cellulose being the major component of lignocellulose, the need for novel and highly efficient cellulases have been realized enormously (Xing et al., 2012).
Cellulases find their use in versatile industrial applications such as in paper industry for deinking of recycled paper, textile industry for biopolishing of fabric and reducing harshness of cotton cloth, laundry industry as an additive to detergents, food and feed industry to improve the digestibility of food, brewing industry and agricultural industry for bioprocessing of crops and many other applications (Bhat, 2000; Xing et al., 2012).
Cellulases belong to glycosyl hydrolase family of enzymes which catalyse cellulolysis in a concerted manner. Endoglucanase (EC 3.2.1.4) randomly cleaves the internal 1,4-P-D-glucan linkage, producing free ends. Exoglucanase (EC 3.2.1.91 and 3.2.1.176) progressively act on reducing and non-reducing ends to release cellobiose. The di-ssacharide produced is then digested by β-glucosidases (EC 3.2.1.21) to release free glucose. These enzymes work synergistically to bring the cellulose hydrolysis (Aubert et al., 1988; Lynd et al., 2002). Endoglucanases are the major enzymes to initiate and bring out extensive hydrolysis of internal linkages. Endoglucanases fall into 14 families of glycosyl hydrolase families, according to the classification by Carbohydrate Active Enzymes database (Lombard et al., 2014)
One of the approaches to discover novel cellulases is through metagenomics which is a culture-independent approach for studying the microbial diversity and exploring novel enzymes of industrial importance (Handelsman, 2004; Zengler et al., 2002).
Among the various natural environments, soil is the most diverse and challenging with respect to the microbiota present in it (Daniel, 2005). Many novel industrially relevant enzymes, like cellulases, amylases, lipases, proteases, xylanases etc. have been discovered from soil metagenomics (Daniel, 2005; Xing et al., 2012). Several of these enzymes have far superior properties w.r.t. activity, specificity, stability etc. than the known enzymes.
Many cellulases have been derived from the metagenomic studies, which have remarkable properties, like thermostability, halostability, pH stability. As examples, novel metagenomic GH5 cellulases have been isolated from ruminal fluid of cow which is active against wide range of substrates (Ko et al., 2013). A thermophillic GH9 endoglucanase having the optimal activity at 75° C. have been isolated from sugarcane bagasse (Kanokratana et al., 2014). A metagenome derived GH12 cellulase isolated from leaf branch compost has the optimum temperature of 90° C. (Okano et al., 2014). The cellulases isolated from rumen of cattle have specific activity ranging from 6-70 U/mg on CMC as substrate (Ferrer et al., 2008). Such properties, associated with newer proteins having industrial relevance is a great need for the full and successful aim of harvesting biomass as sources of affordable and green energy.
Therefore it is an object of the present invention to provide cellulases which are active at high temperature, over wide pH range, broad stability and tolerance to a range of chemical and physical conditions, high activity in the presence of salts and chemicals etc.